Oligonucleotide decoys for the treatment of pain

ABSTRACT

Provided are therapeutic agents such as double-stranded nucleic acids, termed oligonucleotide decoys, pharmaceutical compositions comprising the same, and related methods of modulating nociceptive signaling, for instance, to prevent and/or treat pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 15/504,191, filed Feb. 15, 2017, now U.S. Pat. No. 10,287,583,issued May 14, 2019; which is a 371 of International Application No.PCT/US2015/045268, filed Aug. 14, 2015; which claims priority to U.S.Application No. 62/037,996, filed on Aug. 15, 2014, which isincorporated by reference in its entirety

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ADDY_003_02US_SeqUst_ST25.txt. The text file isabout 12 KB, was created on Mar. 19, 2019, and is being submittedelectronically via EFS-Web.

BACKGROUND Field of the Invention

The present invention relates to therapeutic agents such asdouble-stranded nucleic acids, termed oligonucleotide decoys,pharmaceutical compositions comprising the same, and related methods ofmodulating nociceptive signaling, for instance, to prevent and/or treatpain.

Description of the Related Art

Pain may be defined as an unpleasant sensory and emotional experienceassociated with actual or potential tissue damage, or described in termsof such damage. Chronic pain afflicts at least 40% of the U.S.population and is associated with numerous deleterious medicalconditions. Persistent and highly debilitating, chronic pain isgenerally accompanied by weakness, sleeplessness, a lack of appetite,irritability and depression. Over time, the quality of life isprofoundly affected and patients are often incapable of accomplishingthe simple tasks of everyday life.

Currently used pain treatments apply a three-step pain ladder whichrecommends the administration of drugs as follows: non-opioids (e.g.,aspirin, acetaminophen, etc.), then, as necessary, mild opioids (e.g.,codeine) and finally strong opioids (e.g., morphine). Despite thisarsenal of drugs, over 50% of patients with chronic pain are noteffectively treated.

The ineffectiveness of current pain treatments is, inter alia, due tosignificant toxicity issues with existing drug therapies. Mild to severetoxicity is induced by all classes of pain drugs: non-steroidalinflammatory drugs cause gastro-intestinal damage, coxibs are associatedwith heart failure, and opioids are responsible for numerous sideeffects including respiratory depression, sedation, digestivemalfunctions and addiction.

Transcription factors are important factors in multiple signalingpathways and frequently control the concurrent expression of numerousgenes. Many transcription factors are involved in the regulation of theexpression of genes that are involved in pain including, but not limitedto, BDNF, Transforming Growth factor (TGFB1), CDKN1A, GFAP, POU factors,upstream stimulatory factors (USF1, USF2), EGR1, cAMP-response elementbinding protein/activating transcription factors (CREB/ATF), activatingprotein 1 (AP1), serum response factor (SRF), promoter selectivetranscription factor (SP1), and the runt related transcription factor 1(CBFA2).

Thus, there may be significant therapeutic potential in inhibitingtranscription factors in order to monitor the expression of genesinvolved in pain. Accordingly, what is needed are selective, readilyavailable non-toxic transcription factor inhibitors.

BRIEF SUMMARY

Embodiments of the present invention relate generally to therapeuticagents, such as oligonucleotides, which inhibit the binding of at leastone Krüppel-like family (KLF) transcription factor to its endogenoustranscription factor binding site(s), pharmaceutical compositionscomprising such agents, and related methods of modulating nociceptivesignaling, for example, to prevent and/or treat pain in a subject inneed thereof. In some embodiments, the therapeutic agents aredouble-stranded oligonucleotides (e.g., oligonucleotide decoys), whichcomprise one or more transcription factor binding sites that bind to atleast one KLF transcription factor.

Embodiments of the present invention therefore include oligonucleotidedecoys comprising one or more transcription factor binding sites,wherein the one or more transcription factor binding sites bind to atranscription factor selected from the group consisting of KLF1, KLF2,KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13,KLF14, KLF15, KLF16 and KLF17. In some embodiments, the one or moretranscription factor binding sites bind to one or more transcriptionfactors (1, 2, 3, 4, 5, etc.), selected from one or more of KLF1, KLF2,KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13,KLF14, KLF15, KLF16 and KLF17.

In particular embodiments, the oligonucleotide decoys comprise acombination of at least two transcription factor binding sites, whereineach transcription factor binding site binds to a transcription factorselected from the group consisting of KLF1, KLF2, KLF3, KLF4, KLF5,KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, KLF16and KLF17. In particular embodiments, each transcription factor bindingsite binds to a different KLF transcription factor.

In some embodiments, the oligonucleotide decoy is about 15 to about 35base pairs in length.

In particular embodiments, the oligonucleotide decoy comprises a firsttranscription factor binding site and a second transcription factorbinding site, wherein the first and second transcription binding sitesoverlap. In certain embodiments, the first transcription factor bindingsite binds to KLF9, and the second transcription factor binding sitebinds to KLF15. In certain embodiments, the first transcription factorbinding site binds to KLF9, and the second transcription factor bindingsite binds to KLF6.

In certain embodiments, the oligonucleotide decoy has a firsttranscription factor binding site, a second transcription factor bindingsite, and a third transcription factor binding site, wherein the first,second, and third transcription factor binding sites overlap. Inspecific embodiments, the first transcription factor binding site bindsto KLF6, the second transcription factor binding site binds to KLF9, andthe third transcription factor binding site binds to KLF15.

Certain embodiments relate to one or more population(s) of theoligonucleotide decoys described herein, wherein the population ofoligonucleotide decoys provide a transcription factor binding ratio ofKLF15/KLF9 equal to or less than about 0.8 or equal to or higher thanabout 1.0 in a standard ELISA assay.

Some embodiments relate to population(s) of the oligonucleotide decoys,wherein the population of oligonucleotide decoys provide a totaltranscription factor binding capacity to KLF6 and KLF9 that is equal toor higher than a predetermined value, for instance, an optical densityvalue of about 0.2 OD₄₅₀ in a standard ELISA assay.

In some embodiments, the oligonucleotide decoy (e.g., in the population)comprises a sequence represented by Formula 1 or Formula 2:

(Formula 1; SEQ ID NO: 1)a₁t₂c₃c₄T₅T₆Y₇G₈M₉M₁₀T₁₁Y₁₂Y₁₃K₁₄Y₁₅C₁₆N₁₇H₁₈h₁₉n₂₀n₂₁v₂₂n₂₃n₂₄y₂₅m₂₆h₂₇w₂₈b₂₉v₃₀a₃₁w₃₂ (Formula 2; SEQ ID NO: 2)t₁g₂t₃k₄b₅K₆K₇D₈D₉V₁₀D₁₁N₁₂S₁₃D₁₄N₁₅B₁₆N₁₇N₁₈d₁₉v₂₀m₂₁b₂₂v₂₃m₂₄h₂₅r₂₆m₂₇a₂₈

wherein S is G or C; W is A or T; Y is T or C; D is A, G, or T; B is C,G, or T; K is T or G; M is C or A; H is C, T, or A; V is C, G, or A; Ris A or G; and N is any nucleotide, wherein lower case letters can beeither present or absent, and wherein the numbers in subscript representthe position of a nucleotide in the sequence.

In some embodiments, the oligonucleotide decoy comprises a sequenceselected from the group consisting of SEQ ID NOs:3-35, or a variantthereof. In specific embodiments, the decoy comprises a sequence thathas at least 70% identity with the sequence of SEQ ID NO:28 (16.6.5),SEQ ID NO:25 (16.6.2), SEQ ID NO:19 (17.5), SEQ ID NO:34(T16.6-T17.5Fu1) or SEQ ID NO:35 (T16.6-T17.5 Fu2).

Certain embodiments include an oligonucleotide decoy comprising asequence represented by Formula 1 or Formula 2:

(Formula 1; SEQ ID NO: 1)a₁t₂c₃c₄T₅T₆Y₇G₈M₉M₁₀T₁₁Y₁₂Y₁₃K₁₄Y₁₅C₁₆N₁₇H₁₈h₁₉n₂₀n₂₁v₂₂n₂₃n₂₄y₂₅m₂₆h₂₇w₂₈b₂₉v₃₀a₃₁w₃₂ (Formula 2; SEQ ID NO: 2)t₁g₂t₃k₄b₅K₆K₇D₈D₉V₁₀D₁₁N₁₂S₁₃D₁₄N₁₅B₁₆N₁₇N₁₈d₁₉v₂₀m₂₁b₂₂v₂₃m₂₄h₂₅r₂₆m₂₇a₂₈

wherein S is G or C; W is A or T; Y is T or C; D is A, G, or T; B is C,G, or T; K is T or G; M is C or A; H is C, T, or A; V is C, G, or A; Ris A or G; and N is any nucleotide, wherein lower case letters can beeither present or absent, and wherein the numbers in subscript representthe position of a nucleotide in the sequence.

In some embodiments, the decoy comprises, consists, or consistsessentially of a sequence selected from the group consisting of SEQ IDNOs:3-35, or a variant thereof. In particular embodiments, the decoycomprises a sequence that has at least 70% identity with the sequence ofSEQ ID NO:28 (16.6.5), SEQ ID NO:25 (16.6.2), SEQ ID NO:19 (17.5), SEQID NO:34 (T16.6-T17.5Fu1) or SEQ ID NO:35 (T16.6-T17.5 Fu2).

Also included are pharmaceutical compositions comprising anoligonucleotide decoy or population of decoys described herein and apharmaceutically acceptable carrier. In certain embodiments, theoligonucleotide decoys are provided as salts, hydrates, solvates, orN-oxides derivatives.

Some embodiments include one or more kits comprising an oligonucleotidedecoy or population of decoys described herein, optionally aninstruction for using the oligonucleotide decoy(s).

Also included are methods for modulating the transcription of a genepresent in a cell involved in nociceptive signaling comprisingadministering to the cell an effective amount of an oligonucleotidedecoy or pharmaceutical composition described herein.

Also included are methods for modulating nociceptive signaling in a cellcomprising administering to the cell an effective amount of anoligonucleotide decoy or pharmaceutical composition described herein.

Certain embodiments include methods for preventing and/or treating painin a subject comprising administering to the subject a therapeuticallyeffective amount of an oligonucleotide decoy or pharmaceuticalcomposition described herein. In some embodiments, the pain is a chronicpain. In particular embodiments, the pain is neuropathic pain. In someembodiments, the pain is associated with inflammation. In certainembodiments, the pain is associated with central nervous system orvisceral disorder. In specific embodiments the pain is neuropathic painassociated with inflammation.

Also included are methods for modulating nociceptive signaling in a cellcomprising administering to the cell a therapeutically effective amountof a therapeutic agent, wherein the therapeutic agent inhibits bindingof a transcription factor to its transcription factor binding site,wherein the transcription factor is selected from the group consistingof KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF8, KLF9, KLF10, KLF11, KLF12,KLF13, KLF14, KLF15, KLF16 and KLF17.

In some embodiments, the therapeutic agent provides a binding ratio ofKLF15/KLF9 equal to or less than about 0.8 or equal to or higher thanabout 1.0 in a standard ELISA assay (e.g., based on OD₄₅₀ values orequivalent standard ELISA measurement units). In particular embodiments,the therapeutic agent provides a total transcription factor bindingcapacity to KLF6 and KLF9 that is equal to or higher than an opticaldensity value of about 0.2 OD₄₅₀ in a standard ELISA assay, or acomparable binding level using equivalent standard ELISA measurementunits.

Also included are methods for treating pain in a subject comprisingadministering to the subject a therapeutically effective amount of atherapeutic agent, wherein the therapeutic agent inhibits binding of atranscription factor to its transcription binding site, wherein thetranscription factor is selected from the group consisting of KLF1,KLF2, KLF3, KLF4, KLF5, KLF6, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13,KLF14, KLF15, KLF16 and KLF17. In some embodiments, the therapeuticagent provides a binding ratio of KLF15/KLF9 equal to or less than about0.8 or equal to or higher than about 1.0 in a standard ELISA assay. Incertain embodiments, the therapeutic agent provides a totaltranscription factor binding capacity to KLF6 and KLF9 that is equal toor higher than an optical density value of about 0.2 OD₄₅₀ in a standardELISA assay. In particular embodiments, the pain is neuropathic pain,pain associated with inflammation, and/or neuropathic pain associatedwith inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the KLF binding characteristics of certain of theoligonucleotide decoys, relative to control KLF decoys (highlighted ingray). Binding values to KLF6, KLF9, and KLF15 are presented as mean andSEM OD₄₅₀ values from the in vitro ELISA binding assay described inExample 1. The corresponding N is also listed. The efficacy for treatingneuropathic and/or neuro-inflammatory pain is presented as percentage(%) of pain reduction relative to control during the testing period(^(˜)4-8 weeks total, ^(˜)2-4 weeks following treatment depending on thestudy) of the corresponding animal studies. N/A=Non-applicable.

FIGS. 2A-B show the efficacy of certain of the oligonucleotide decoys inthe spared nerve injury (SNI) model of chronic neuropathic pain. Painwas measured as mechanical hypersensitivity using repetitive von Freyfilaments. Oligonucleotide decoys (200 nmoles) or vehicle were injectedonce intrathecally at post-operative day 14 (POD14). Mean+SEM values oftotal responses to von Frey stimulations were normalized on the baselinepain values measured at POD14 prior to the injection of vehicle ordecoys; pre-injection data before POD14 are combined across groups,T-test vs. vehicle at a given time-point: *p≤0.05, decoy vs. vehicledata distribution post-treatment (POD 17-POD31): p≤0.001 for TFD16,16.6.2, 16.6.5, TFD17, 17.5, p=0.005 for 17.1, p=0.39 for 16.9 andp=0.46 for 17.9; n=4 rats per testing group. The X-axis showspost-operative days (POD).

FIGS. 3A-B show the efficacy of certain of the oligonucleotide decoys inthe chronic constriction injury (CCI) model of chronicneuro-inflammatory pain. Pain was measured as mechanicalhypersensitivity using repetitive von Frey filaments. Oligonucleotidedecoys (200 nmoles) or vehicle were injected once intrathecally atpost-operative day 14 (POD14). Mean+SEM values of total responses to vonFrey stimulations were normalized on the baseline pain values measuredat POD14 prior to the injection of vehicle or decoys; pre-injection databefore POD14 are combined across groups, T-test vs. vehicle at a giventime-point: *p≤0.1, **p≤0.05, decoy vs. vehicle data distributionpost-treatment (POD 17-POD31): p=0.23 for TFD16, p=0.01 for 16.6.2,p=0.03 for 16.6.5, 0.02 for 16.9, p=0.0004 for TFD17, p=0.005 for 17.1,p=0.004 for 17.5 and p=0.12 for 17.9; n=4 rats per testing group (except17.9: n=3 due to 1 rat exclusion due to insufficient baseline pain valueat POD14). The X-axis shows post-operative days (POD).

FIG. 4A-C show the efficacy level of certain of the oligonucleotidedecoys in relation to their ratio of KLF15/KLF9 binding (4A),coefficients of linear correlation between the efficacy for treatingchronic neuropathic pain and the binding parameters to KLF6, KLF9 andKLF15 (4B), and a linear regression of efficacy levels for thepopulation of the tested decoys in relation to their KLF15/KLF9 bindingratios, excluding ratios 0.9 (4C). The efficacy level of each decoy wasmeasured as the percentage of pain relief vs. control in the SNI modelof chronic pain during the testing period (^(˜)4-8 weeks total, ^(˜)2-4weeks following treatment depending on the study).

FIGS. 5A-C show the efficacy level of certain of the oligonucleotidedecoys in relation to their combined binding to KLF6, KLF9 and KLF15(5A), coefficients of linear correlation between the efficacy fortreating chronic neuro-inflammatory pain and the binding parameters toKLF6, KLF9 and KLF15 (5B), and a linear regression of efficacy level forthe population of tested decoys in relation to their total bindingcapacity to KLF6 and KLF9, as indicated by their 1/(KLF6+KLF9) bindingratios (5C). The efficacy level of each decoy was measured as thepercentage of pain reduction vs. control in the CCI model of chronicpain during the testing period (^(˜)4-8 weeks total, 2-4 weeks followingtreatment depending on the study).

FIG. 6 shows the differential pattern of efficacy of certain of theoligonucleotide decoys (white lozenges) relative to control KLF decoysfrom the literature (TFDC1, TFDC2, and TFD3, which contains twoKLF-consensus CACCC-box binding sites, black lozenges), acrosscomplementary etiologies of pain, from neuropathic (Y-axis) to painincluding inflammatory components (X-axis).

FIG. 7 shows a plot of the 1/(KLF6+KLF9) binding ratio (diamonds), whichis indicative of the efficacy for treating neuro-inflammatory pain (thelower, the more efficacy), and of the KLF15/KLF9 binding ratio(squares), which is indicative of the efficacy for treating neuropathicpain (the higher, the more efficacy), for the oligonucleotide decoys ofthe invention in Table 2 (X-axis, KLF decoys: 1=16.5, 2=16.6.7, 3=17.7,4=17.1, 5=16.2, 6=16.6.2, 7=17.3, 8=16.6, 9=17.9, 10=17.5, 11=16.8,12=16.9, 13=17.8, 14=17.4, 15=17.1, 16=16.4, 17=16.1, 18=17.2, 19=16.0,20=17.5.3, 21=16.6.3, 22=17.5.1, 23=16.3, 24=16.6.5, 25=16.10, 26=17.6,27=T16.6-T17.5 Fu2, 28=17.0, 29=16.6.4, 30=16.6.6, 31=17.5.2, 32=16.7,T16.6-T17.5 Fu1 not listed due to non-applicable values).

FIGS. 8A-B show the effect of ascending dose levels of 16.6.5oligonucleotide decoy in the SNI model of chronic neuropathic pain (A)and in the CCI model of chronic neuro-inflammatory pain (B). Pain wasmeasured as mechanical hypersensitivity using repetitive von Freyfilaments. 16.6.5 or vehicle were injected once intrathecally atpost-operative day 14 (POD14). Mean+SEM values of total responses to vonFrey stimulations were normalized on the baseline pain values measuredat POD14 prior to the injection of vehicle or decoys; pre-injection databefore POD14 were combined across groups, T-test vs. vehicle at a giventime point: *p≤0.05, 16.6.5 vs. vehicle data distribution post-treatment(POD 17-POD31): p≤0.001 for 100, 200 and 300 nmoles dose-levels in theSNI model, p=0.02 for 200 moles and p≤0.001 for 300 nmoles dose-levelsin the CCI model; n=4 rats per testing group. The X-axis showspost-operative days (POD).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight, or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight, or length.

“Binding,” as used in the context of transcription factors binding totherapeutic agents such as oligonucleotide decoys, refers to a directinteraction (e.g., non-covalent bonding between the transcription factorand the oligonucleotide decoy, including hydrogen-bonding, van der Waalsbonding, etc.) between a transcription factor and an oligonucleotidedecoy. Accordingly, a therapeutic agent such as an oligonucleotide thatdoes not bind to a transcription factor does not directly interact withsaid transcription factor, and vice versa.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of:” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

“Chronic” refers to a period of time comprising months (e.g., at leasttwo months) or years.

“Homology” refers to the percentage number of nucleotides that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as EMBOSS PairwiseAlignment Algorithm (available from the European BioinformaticsInstitute (EBI)), the ClustalW program (also available from the EuropeanBioinformatics Institute (EBI)), or the BLAST program (BLAST Manual,Altschul et al., Natl Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLMNIH), Bethesda, Md., and Altschul et al., (1997) NAR 25:3389 3402), orGAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In thisway sequences of a similar or substantially different length to thosecited herein could be compared by insertion of gaps into the alignment,such gaps being determined, for example, by the comparison algorithmused by GAP.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide” or “isolated oligonucleotide,” asused herein, may refer to a polynucleotide that has been purified orremoved from the sequences that flank it in a naturally-occurring state,e.g., a DNA fragment that is removed from the sequences that areadjacent to the fragment in the genome. The term “isolating” as itrelates to cells refers to the purification of cells (e.g., fibroblasts,lymphoblasts) from a source subject (e.g., a subject with apolynucleotide repeat disease). In the context of mRNA or protein,“isolating” refers to the recovery of mRNA or protein from a source,e.g., cells.

The term “modulate” includes an “increase” or “decrease” an one or morequantifiable parameters, optionally by a defined and/or statisticallysignificant amount. By “increase” or “increasing,” “enhance” or“enhancing,” or “stimulate” or “stimulating,” refers generally to theability of one or more agents such as oligonucleotide decoys to produceor cause a greater physiological or cellular response in a cell or asubject, such as the activity of a transcription factor (e.g., geneexpression), relative to the response caused by either no agent or acontrol compound. Relevant physiological or cellular responses (in vivoor in vitro) will be apparent to persons skilled in the art. An“increased” or “enhanced” amount or response may be “statisticallysignificant” relative to an amount or response produced by no agent or acontrol composition, and may include an increase that is 1.1, 1.2, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000times) (including all integers and ranges between and above 1, e.g.,1.5, 1.6, 1.7, 1.8) the amount or response produced by either no agentor a control compound. The term “reduce” or “inhibit” may relategenerally to the ability of one or more agents such as oligonucleotidedecoys to “decrease” a relevant physiological or cellular response in acell or a subject, such as the activity of a transcription factor (e.g.,gene expression), a physiological process (e.g., nociceptive signaling),or a symptom of a disease or condition described herein (e.g., pain),relative to the response caused by either no agent or a controlcompound. Relevant physiological or cellular responses (in vivo or invitro) will be apparent to persons skilled in the art and can bemeasured according to routine techniques. A “decrease” in a response maybe “statistically significant” as compared to the response produced byno agent or a control composition, and may include a 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% decrease, including all integers and ranges in between.

“Modulation of gene expression level” includes any change in geneexpression level, including an induction or activation (e.g., anincrease in gene expression), an inhibition or suppression (e.g., adecrease in gene expression), or a stabilization (e.g., prevention ofthe up-regulation or down-regulation of a gene that ordinarily occurs inresponse to a stimulus, such as a pain-inducing stimulus).

“Nociceptive signaling” refers to molecular and cellular mechanismsinvolved in the detection of a noxious stimulus or of a potentiallyharmful stimulus, which leads to the perception of pain. Particularexamples include neurotransmitter synthesis and release,neurotransmitter-induced signaling, membrane depolarization, and relatedintra-cellular and inter-cellular signaling events.

“Pain” refers to an unpleasant sensory and emotional experience that isassociated with actual or potential tissue damage or described in suchterms. All of the different manifestations and qualities of pain,including mechanical pain (e.g., induced by a mechanical stimulus or bybody motion), temperature-induced pain (e.g., pain induced by hot, warmand/or cold temperatures), and chemically-induced pain (e.g., paininduced by a chemical). In certain embodiments, pain is chronic,sub-chronic, acute, or sub-acute. In certain embodiments, pain featureshyperalgesia (e.g., an increased sensitivity to a painful stimulus)and/or allodynia (e.g., a painful response to a usually non-painfulstimulus). In certain embodiments, pain is pre-existing in a patient. Inother embodiments, pain is iatrogenic, induced in a patient (e.g.,post-operative pain).

“Preventing” or “prevention” includes (1) a reduction in the risk ofacquiring a disease or disorder (e.g., causing at least one of theclinical symptoms of a disease not to develop in a patient that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease), and/or (2) a reduction in the likelyseverity of a symptom associated with a disease or disorder (e.g.,reducing the likely severity of at least one of the clinical symptoms ofa disease in a patient that may be exposed to or predisposed to thedisease but does not yet experience or display symptoms of the disease).

The terms “sequence identity” or, for example, comprising a “sequence50% identical to,” as used herein, refer to the extent that sequencesare identical on a nucleotide-by-nucleotide basis over a window ofcomparison. Thus, a “percentage of sequence identity” may be calculatedby comparing two optimally aligned sequences over the window ofcomparison, determining the number of positions at which the identicalnucleic acid base (e.g., A, T, C, or G) occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. In some embodiments, optimal alignmentof sequences for aligning a comparison window may be conducted by usingthe EMBOSS Pairwise Alignment Algorithm (available from the EuropeanBioinformatics Institute (EBI)), the ClustalW program (also availablefrom the European Bioinformatics Institute (EBI)), or the BLAST program(BLAST Manual, Altschul et al., Natl Cent. Biotechnol. Inf., Natl Lib.Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., (1997) NAR25:3389 3402). In certain embodiments, the alignment of sequences foraligning a comparison window is conducted against the entire length ofthe reference sequence (e.g., from the Sequence Listing). In someembodiments, the alignment of sequences for aligning a comparison windowis conducted against a portion of the reference sequence, for example,about, at least about, or no more than about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70,80, 90, or 100 contiguous nucleotides of the reference sequence.

A “subject” or a “subject in need thereof” or a “patient” includes amammalian subject such as a primate or human subject.

“Sub-acute” refers to a period of time comprising hours (e.g., 1-24hours, including all integers and ranges in between).

“Sub-chronic” refers to a period of time comprising days or months(e.g., less than two or three months).

“Treating” or “treatment” of any disease or disorder refers, in someembodiments, to ameliorating the disease or disorder (e.g., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In some embodiments, “treating” or “treatment” refersto ameliorating at least one physical and/or biological parameter, whichmay not be discernible by the patient. In certain embodiments,“treating” or “treatment” refers to inhibiting the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter) or both.In some embodiments, “treating” or “treatment” refers to delaying theonset of the disease or disorder. “Treatment” or “prophylaxis” does notnecessarily indicate complete eradication, cure, or prevention of thedisease or condition, or associated symptoms thereof.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient, is sufficient to effect such treatmentof a particular disease or condition. The “therapeutically effectiveamount” will vary depending on the compound, the disease, the severityof the disease, and the age, weight, etc., of the patient to be treated.

Oligonucleotide Decoys and Other Therapeutic Agents

Embodiments of the present invention relate generally to therapeuticagents that inhibit binding of at least one transcription factor to atleast one of its (endogenous) transcription binding site. Particularexamples include oligonucleotide decoys that comprise one or moretranscription binding sites that bind to at least one transcriptionfactor, and thereby alter the ability of the transcription factor(s) tomodulate gene expression. In certain embodiments, the transcriptionfactor is one or more members of the Krüppel-like family (KLFs) oftranscription factors, examples of which include KLF1, KLF2, KLF3, KLF4,KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15,KLF16 and KLF17.

Thus, certain embodiments include an oligonucleotide decoy thatcomprises one or more (e.g., 1, 2, 3, 4, 5, etc.) transcription factorbinding sites, where the one or more transcription factor binding sitebinds to a transcription factor selected from the group consisting ofKLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11,KLF12, KLF13, KLF14, KLF15, KLF16 and KLF17.

Also included are oligonucleotide decoys that comprise a combination ofat least two (e.g., 2, 3, 4, 5, etc.) transcription factor bindingsites, wherein each transcription factor binding site binds to atranscription factor selected from the group consisting of KLF1, KLF2,KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13,KLF14, KLF15, KLF16 and KLF17. Particular examples of combinations oftranscription factor binding sites include those that bind to KLF6/KLF9,KLF9/KLF15, or KLF6/KLF9/KLF15.

The term “oligonucleotide” includes any double-stranded or substantiallydouble-stranded, nucleic acid-containing polymer generally less thanapproximately 200 nucleotides (or 100 base pairs) and including, but notlimited to, DNA, RNA and RNA-DNA hybrids.

In some embodiments, the oligonucleotide is about, at least about, or nomore than about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, or 200 nucleotides in length (includingall integers and ranges in between), and optionally comprises about, atleast about, or no more than about, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 base-pairednucleotides (including all integers and ranges in between). Inparticular embodiments, the oligonucleotide decoy is about 15 to about35 base pairs in length.

In some embodiments, the oligonucleotide decoy comprises a firsttranscription factor binding site and a second transcription bindingsite, optionally wherein the first transcription binding site and thesecond transcription binding site overlap. In specific embodiments, thefirst transcription factor binding site binds to KLF9 and the secondtranscription factor binding site binds to KLF15. In particularembodiments, the first transcription factor binding site binds to KLF9and the second transcription factor binding site binds to KLF6.

Also included are oligonucleotide decoys that have a first transcriptionfactor binding site, a second transcription factor binding site, and athird transcription factor binding site, optionally wherein the first,second, and third transcription binding sites overlap. In specificembodiments, the first transcription factor binding site binds to KLF6,the second transcription factor binding site binds to KLF9, and thethird transcription factor binding site binds to KLF15.

In certain embodiments, the oligonucleotide decoy (e.g., the sensestrand of the decoy) comprises, consists, or consists essentially of asequence (e.g., double-stranded sequence) represented by Formula 1 orFormula 2, shown in Table 1 below, or a variant thereof, or a complementthereof (e.g., the antisense sequence).

TABLE 1 Sequence SEQ name Sequence (5′ to 3′) ID NO: Formula 1a₁t₂c₃c₄T₅T₆Y₇G₈M₉M₁₀T₁₁Y₁₂Y₁₃K₁₄Y₁₅C₁₆N₁₇H₁₈h₁₉n₂₀n₂₁v₂₂n₂₃ 1n₂₄y₂₅m₂₆h₂₇w₂₈b₂₉v₃₀a₃₁w₃₂ Formula 2t₁g₂t₃k₄b₅K₆K₇D₈D₉V₁₀D₁₁N₁₂S₁₃D₁₄N₁₅B₁₆N₁₇N₁₈d₁₉v₂₀m₂₁b₂₂v₂₃ 2m₂₄h₂₅r₂₆m₂₇a₂₈ wherein S is G or C; W is A or T; Y is T or C; D is A,G, or T; B is C, G, or T; K is T or G; M is C or A; H is C, T, or A; Vis C, G, or A; R is A or G; and N is any nucleotide, wherein lower caseletters can be either present or absent, and wherein the numbers insubscript represent the position of a nucleotide in the sequence.

In specific embodiments, the oligonucleotide decoy (e.g., the sensestrand of the decoy) comprises, consists, or consists essentially of asequence in Table 2 below, or a variant thereof, or a complement thereof(e.g., the antisense sequence).

TABLE 2 SEQ Sequence ID name Sequence (5′ to 3′) NO: 16.0TTTGCCTCCTTCGATCCC  3 16.1 ATCCTTTGCCTCCTTCGA  4 16.2ATCCTTTGCCTCCTTCCCTTTGCCTCCTTCAA  5 16.3 CCTTTGCCTCCTTCCCTTTGCCTCCTTC  616.4 ATCCTTTGCCTCCTTCGAAGGAGGCAAAGGAT  7 16.5ATCCTTTGCCTCCTTCCTTTGCCTCCTTCAA  8 16.6 ATCCTTTGCCTCCTTCGCCTCCTTCAA  916.7 CCTTTGCCTCCTTCGCCTCCTTC 10 16.8 ATCCTTTGCCTCCTTCTCCTTCAA 11 16.9ATCCTTTGCCTTTGCCTCCTTCAA 12 16.10 CCTTTGCCTTTGCCTCCTTC 13 17.0TGTTTGGGAGAGCTT 14 17.1 GCTTTGGGAGGATAC 15 17.2 TGGGAGAGCTTTGGGA 16 17.3TGTTTGGGAGATTTGGGAGGATAC 17 17.4 TTTGGGAGATTTGGGAGGAT 18 17.5TGTTTGGGAGAATCCTCCCAAAGC 19 17.6 TTTGGGAGAATCCTCCCAAA 20 17.7TGTTTGGGAGAGCTATCCTCCCAAAGC 21 17.8 TTTGGGAGAGCTATCCTCCCAAA 22 17.9TGTTTGGGAGAGGGAGGATAC 23 17.10 TGTTTGGGTTTGGGAGGATAC 24 16.6.2CCTTTGCCTCCTTCGCCTCCTTCAA 25 16.6.3 TCCTTTGCCTCCTTCGCCTCCTTCA 26 16.6.4CCTTTGCCTCCTTCGCCTCCTTCA 27 16.6.5 ATCCTTCGCCTCCTTCAA 28 16.6.6ATCCTTCGCCTTCGCCTCCTTCAA 29 16.6.7 ATCCTTCGCCTCCTTCGCCTCCTTCAA 30 17.5.1TGTTTGGGAGAATCCTCCCAAA 31 17.5.2 TTTGGGAGAATCCTCCCAAAGC 32 17.5.3GTTTGGGAGAATCCTCCCAAAG 33 T16.6- ATCCTTCGCCTCCTTCTCCCAAAGC 34 T17.5 Fu1T16.6- ATCCTTCGAATCCTTCCAAAGC 35 T17.5 Fu2

In the formulas and sequences described herein, “A” is an adeninenucleotide, “C” is a cytosine nucleotide, “G” is a guanine nucleotide,“T” is a thymine nucleotide, and “N” can be any nucleotide, preferablyA, C, G, or T. Although the formulas and sequences show a single strand,it should be understood that a complementary antisense strand isincluded as part of the structure of the oligonucleotide decoys. Incertain embodiments, any one or more “T” can be a “U” or uracilnucleotide.

Certain oligonucleotide decoys thus comprise, consist, or consistessentially of a sequence in Table 1 or Table 2 (e.g., SEQ ID NOS:1-35)or a variant or contiguous or non-contiguous portion(s) thereof. Forinstance, certain oligonucleotide decoys comprise about or at leastabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous or non-contiguousnucleotides of any of the targeting sequences in Table 1 or Table 2(e.g., SEQ ID NOS:1-35), and which bind to one or more KLF transcriptionfactors described herein (e.g., KLF1, KLF2, KLF3, KLF4, KLF5, KLF6,KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, KLF16,KLF17). For non-contiguous portions, intervening nucleotides can bedeleted or substituted with a different nucleotide, or interveningnucleotides can be added. Additional examples of variants includeoligonucleotide decoys having at least or at least about 70% sequenceidentity or homology (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or homology)to the entire length or a contiguous portion of a sequence in Table 1 orTable 2 (e.g., SEQ ID NOS:1-35), and which bind to one or more KLFtranscription factors described herein (e.g., KLF1, KLF2, KLF3, KLF4,KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15,KLF16, KLF17). In some embodiments, the contiguous portion is about, atleast about, or no more than about 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90,or 100 contiguous nucleotides of a sequence in Table 1 or Table 2 (e.g.,SEQ ID NOS:1-35).

An oligonucleotide decoy having a certain percent (e.g., 65%, 70%, 75%,80%, 85%, 90%, 95%, or 99%) of sequence identity with another sequencemeans that, when aligned, that percentage determines the level ofcorrespondence of bases arrangement in comparing the two sequences. Thisalignment and the percent homology or identity can be determined usingany suitable software program known in the art that allows localalignment. In some embodiments, such programs include but are notlimited to the EMBOSS Pairwise Alignment Algorithm (available from theEuropean Bioinformatics Institute (EBI)), the ClustalW program (alsoavailable from the European Bioinformatics Institute (EBI)), or theBLAST program (BLAST Manual, Altschul et al., Natl Cent. Biotechnol.Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al.,(1997) NAR 25:3389 3402).

As noted above, one skilled in the art will recognize that the sequencesencompassed by the invention include those that are fully or partiallycomplementary to the sequences described herein, including those thathybridize under stringent hybridization conditions with an exemplifiedsequence (e.g., Tables 1 and 2; SEQ ID NOs:1-35). A nucleic acid ishybridizable to another nucleic acid when a single stranded form of thenucleic acid can anneal to the other single stranded nucleic acid underappropriate conditions of temperature and solution ionic strength.Hybridization conditions are well known in the art. In some embodiments,annealing may occur during a slow decrease of temperature from adenaturizing temperature (e.g., 100° C.) to room temperature in a saltcontaining solvent (e.g., Tris-EDTA buffer).

Also included are populations of oligonucleotide decoys, including thosewhich provide a transcription factor binding ratio to a combination ofKLF transcription factors (e.g., KLF15/KLF9), and/or a totaltranscription binding capacity to one or more (e.g., 1, 2, 3, 4, 5,etc.) KLF transcription factors (e.g., KLF6+KLF9), which is definedrelative to a predetermined amount. In some embodiments, thetranscription factor binding ratio or total transcription bindingcapacity is about equal to, less than, or higher than a predeterminedlevel or amount. A “predetermined level” for defining a relative bindingratio or a total binding capacity can be established using a variety oftechniques, such as standard ELISA assays (see the Examples).

For example, in specific embodiments, the population of oligonucleotidedecoys provides a transcription factor binding ratio of KLF15/KLF9 thatis equal to or less than about 0.8 or equal to or higher than about 1.0,based on OD₄₅₀ values (or equivalent standard ELISA measurement units,e.g., fluorescence) from a standard ELISA assay (see the Examples). Inspecific embodiments, the transcription factor binding ratio ofKLF15/KLF9 is equal to or less than about 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, 0.01 or less (including all ranges and integers inbetween) based on OD₄₅₀ values from a standard ELISA assay. In someembodiments, the transcription factor binding ratio of KLF15/KLF9 isequal to or higher than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or10.0, or higher (including all ranges and integers in between) based onOD₄₅₀ values (or equivalent standard ELISA measurement units) from astandard ELISA assay.

In some embodiments, the population of oligonucleotide decoys provides atotal transcription factor binding capacity to KLF6 and KLF9 that isequal to or higher than a predetermined amount. In some instances, thepredetermined amount is an optical density value (or an equivalentstandard ELISA measurement unit, e.g., fluorescence) of about 0.2 OD₄₅₀or higher as measured in a standard ELISA assay (see the Examples). Insome embodiments, the predetermined amount or the total transcriptionfactor binding capacity to KLF6 and KLF9 is equal to or higher thanabout 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or higher (including all ranges andintegers in between) based on OD₄₅₀ values (or equivalent standard ELISAmeasurement units) from a standard ELISA assay.

In some embodiments, the population of oligonucleotide decoys provides atotal transcription factor binding capacity to KLF6 and KLF9 that isequal to or less than a predetermined amount. In some embodiments, thepredetermined amount or the total transcription factor binding capacityto KLF6 and KLF9 is indicated as 1/(KLF6+KLF9) based on an opticaldensity value (or an equivalent standard ELISA measurement unit, e.g.,fluorescence) from a standard ELISA assay (see the Examples). Forinstance, in particular embodiments, the total transcription factorbinding capacity to KLF6 and KLF9 (as indicated by 1/(KLF6+KLF9) isabout 5 or less based on OD₄₅₀ values (or an equivalent standard ELISAmeasurement unit) from a standard ELISA assay. In some embodiments, thetotal transcription factor binding capacity to KLF6 and KLF9 (asindicated by 1/(KLF6+KLF9)) is equal to or less than about 5, 4, 3, 2,1, 0.5, 0.1, or less (including all ranges and integers in between)based on OD₄₅₀ values (or an equivalent standard ELISA measurement unit)from a standard ELISA assay.

The population of oligonucleotide decoys can be composed of oneoligonucleotide decoy, or a combination of two or more (e.g., 2, 3, 4,5, etc.) oligonucleotide decoys. In certain embodiments, the populationof oligonucleotide decoys is composed of one oligonucleotide decoy witha single KLF transcription factor binding site. In some embodiments, thepopulation of oligonucleotide decoys is composed of one oligonucleotidedecoy with combination of at least two (e.g., 2, 3, 4, 5, etc.)transcription factor binding sites, which bind to the same or different(e.g., two or at least two different) KLF transcription factors. In someembodiments, the population of oligonucleotide decoys comprises oneoligonucleotide decoy with combination of at least three (e.g., 3, 4, 5,etc.) transcription factor binding sites, which bind to the same ordifferent (e.g., three or at least three different) KLF transcriptionfactors. Other combinations will be apparent to persons skilled in theart.

Generally, the oligonucleotide decoys disclosed herein may be used tobind and, e.g., thereby inhibit, transcription factors that modulate theexpression of genes involved nociceptive signaling and/or a subject's(e.g., patient's) perception of pain. An oligonucleotide decoy that isdesigned to bind to a specific transcription factor has a nucleic acidsequence mimicking the endogenous genomic DNA sequence normally bound bythe transcription factor. Accordingly, in some aspects theoligonucleotide decoys disclosed herein inhibit a necessary step forgene expression and regulation. Further, the oligonucleotide decoysdisclosed herein may bind to one or a number of different transcriptionfactors.

The term oligonucleotide encompasses sequences that include any of theknown base analogs of DNA and RNA including, but not limited to,2,6-diaminopurine, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,uracil-5-oxyacetic acid, N6-isopentenyladenine, 1-methyladenine,N-uracil-5-oxyacetic acid methylester, queosine, 2-thiocytosine,5-bromouracil, methylphosphonate, phosphorodithioate, ormacetal,3′-thioformacetal, nitroxide backbone, sulfone, sulfamate, morpholinoderivatives, locked nucleic acid (LNA) derivatives, and/or peptidenucleic acid (PNA) derivatives. In some embodiments, the oligonucleotideis composed of two complementary single-stranded oligonucleotides thatare annealed together. In some embodiments, the oligonucleotide iscomposed of one single-stranded oligonucleotide that formsintramolecular base pairs to create a substantially double-strandedstructure.

In some embodiments, the oligonucleotide decoys disclosed herein arechemically modified by methods well known to the skilled artisan (e.g.,incorporation of phosphorothioate, methylphosphonate,phosphorodithioate, phosphoramidates, carbonate, thioether, siloxane,acetamidate or carboxymethyl ester linkages between nucleotides), forexample, to prevent degradation by nucleases within cells and/or inextra-cellular fluids (e.g., serum, cerebrospinal fluid). In someembodiments, the oligonucleotide decoys are designed to form hairpin anddumbbell structures, which can also prevent or hinder nucleasedegradation. In particular embodiments, the oligonucleotide decoys areinserted as a portion of a larger plasmid capable of episomalmaintenance or constitutive replication in the target cell in order toprovide longer-term, enhanced intracellular exposure to the decoysequence and/or reduce its degradation. Accordingly, any chemicalmodification or structural alteration known in the art to enhanceoligonucleotide stability is within the scope of the present disclosure.In some embodiments, the oligonucleotide decoys disclosed herein may beattached, for example, to polyethylene glycol polymers, peptides (e.g.,a protein translocation domain) or proteins which improve thetherapeutic effect of oligonucleotide decoys. Such modifiedoligonucleotide decoys may preferentially traverse the cell membrane.

The oligonucleotide decoys described herein may generally be utilized asthe free acid or free base. Alternatively, the oligonucleotide decoysmay be used in the form of acid or base addition salts. Acid additionsalts of the free amino compounds of the present invention may beprepared by methods well known in the art, and may be formed fromorganic and inorganic acids. Suitable organic acids include maleic,fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids.

Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric,phosphoric, and nitric acids. Base addition salts included those saltsthat form with the carboxylate anion and include salts formed withorganic and inorganic cations such as those chosen from the alkali andalkaline earth metals (for example, lithium, sodium, calcium, potassium,magnesium, barium and calcium), as well as the ammonium ion andsubstituted derivatives thereof (e.g., dibenzylammonium, benzylammonium,2-hydroxyethylammonium, and the like). Thus, the term “pharmaceuticallyacceptable salt” is intended to encompass any and all acceptable saltforms.

Prodrugs are also included. Prodrugs are any covalently bonded carriersthat release a compound in vivo when such prodrug is administered to apatient. Prodrugs are generally prepared by modifying functional groupsin a way such that the modification is cleaved, either by routinemanipulation or in vivo, yielding the parent compound. Prodrugs include,for example, compounds of this invention wherein hydroxy, amine orsulfhydryl groups are bonded to any group that, when administered to apatient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus,representative examples of prodrugs include (but are not limited to)acetate, formate and benzoate derivatives of alcohol and aminefunctional groups of the oligonucleotide decoys described herein.Further, in the case of a carboxylic acid (—COOH), esters may beemployed, such as methyl esters, ethyl esters, and the like.

In certain embodiments, the oligonucleotide decoys are provided assalts, hydrates, solvates, or N-oxide derivatives. In certainembodiments, the oligonucleotide decoys are provided in solution (e.g.,a saline solution having a physiologic pH) or in lyophilized form. Insome embodiments, the oligonucleotide decoys are provided in liposomes.

The oligonucleotide decoys described herein may be made by conventionalmethods known in the art and thus are well within the knowledge of theskilled artisan. The activity of oligonucleotide decoys and variantsthereof can be assayed according to routine techniques in the art (seethe Examples). In particular embodiments, the oligonucleotide decoy is asynthetic oligonucleotide (i.e., a chemically-synthesized,non-naturally-occurring oligonucleotide).

Also included are non-oligonucleotide-based therapeutic agents,including those that inhibit binding of a transcription factor to itsendogenous transcription binding site, for instance, by specificallybinding to a KLF transcription factor, or by specifically binding to itsendogenous transcription factor binding site (e.g., by mimicking the KLFtranscription factor binding site). Examples of therapeutic agentsinclude binding agents such as antibodies, small molecules, peptides,adnectins, anticalins, Darpins, anaphones, and aptamers, which exhibitbinding specificity for a KLF transcription factor, e.g., a KLF factortranscription factor binding site domain, or which exhibit bindingspecificity for an endogenous KLF transcription factor binding site.

A binding agent is said to “exhibit binding specificity for,”“specifically bind to,” a KLF polypeptide (e.g., a transcription factorbinding domain thereof), or an endogenous KLF transcription factorbinding site (e.g., double-stranded DNA sequence), if it reacts at adetectable level (within, for example, an ELISA assay) with thepolypeptide or nucleic acid, and does not react detectably in asignificant (e.g., statistically significant) manner with unrelatedstructures under similar conditions.

The term “antibody” relates to an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antigen-binding domain. CDR grafted antibodies arealso contemplated by this term. The term “antigen-binding portion of anantibody,” “antigen-binding fragment,” “antigen-binding domain,”“antibody fragment” or a “functional fragment of an antibody” are usedinterchangeably in the present invention to include one or morefragments of an antibody that retain the ability to specifically bind toan antigen (see, e.g., Holliger et al., Nature Biotech. 23 (9):1126-1129 (2005)).

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for a polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Also included aremethods that utilize transgenic animals such as mice to express humanantibodies. See, e.g., Neuberger et al., Nature Biotechnology 14:826,1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101,1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995.Particular examples include the VELOCIMMUNE® platform by REGENEREX®(see, e.g., U.S. Pat. No. 6,596,541). Antibodies can also be generatedor identified by the use of phage display or yeast display libraries(see, e.g., U.S. Pat. No. 7,244,592; Chao et al., Nature Protocols.1:755-768, 2006).

As noted above, “peptides” that inhibit binding of a KLF transcriptionfactor to its transcription factor binding site are included as bindingagents. The term peptide typically refers to a polymer of amino acidresidues and to variants and synthetic analogues of the same. In certainembodiments, the term “peptide” refers to relatively short polypeptides,including peptides that consist of about 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids,including all integers and ranges (e.g., 5-10, 8-12, 10-15, 15-20,20-25, 25-30, 30-40, 40-50) in between, and which, for example, bind toone or more regions of a KLF transcription factor, e.g., a transcriptionfactor binding domain, or mimic the KLF transcription factor by bindingto at least one of its endogenous transcription factor binding sites.Peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids.

As noted above, the present invention includes small molecules thatinhibit binding of a KLF transcription factor to its transcriptionfactor binding site. A “small molecule” refers to an organic orinorganic compound that is of synthetic or biological origin, but istypically not a polymer. Organic compounds include a large class ofchemical compounds whose molecules contain carbon, typically excludingthose that contain only carbonates, simple oxides of carbon, orcyanides. A “polymer” refers generally to a large molecule ormacromolecule composed of repeating structural units, which aretypically connected by covalent chemical bond. In certain embodiments, asmall molecule has a molecular weight of less than 1000-2000 Daltons,typically between about 300 and 700 Daltons, and including about 50,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750,700, 850, 800, 950, 1000 or 2000 Daltons.

Aptamers that inhibit binding of a KLF transcription factor to itstranscription factor binding site are also included as binding agents(see, e.g., Ellington et al., Nature. 346, 818-22, 1990; and Tuerk etal., Science. 249, 505-10, 1990). Examples of aptamers included nucleicacid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers.Nucleic acid aptamers refer generally to nucleic acid species withsecondary and tertiary structures that have been engineered throughrepeated rounds of in vitro selection or equivalent method, such asSELEX (systematic evolution of ligands by exponential enrichment), tobind to various molecular targets such as small molecules, proteins,nucleic acids, and even cells, tissues and organisms. See, e.g., U.S.Pat. Nos. 6,376,190; and 6,387,620 Hence, included are nucleic acidaptamers that bind to one or more regions of a KLF transcription factor,e.g., a transcription factor binding domain, or which bind to at leastone of its endogenous transcription factor binding sites.

Peptide aptamers typically include a variable peptide loop attached atboth ends to a protein scaffold, a double structural constraint thattypically increases the binding affinity of the peptide aptamer tolevels comparable to that of an antibody's (e.g., in the nanomolarrange). In certain embodiments, the variable loop length may be composedof about 10-20 amino acids (including all integers in between), and thescaffold may include any protein that has good solubility and compacityproperties. Certain exemplary embodiments may utilize the bacterialprotein Thioredoxin-A as a scaffold protein, the variable loop beinginserted within the reducing active site (-Cys-Gly-Pro-Cys-loop in thewild protein), with the two cysteine lateral chains being able to form adisulfide bridge. Methods for identifying peptide aptamers aredescribed, for example, in U.S. Application No. 2003/0108532. Hence,included are peptide aptamers that bind to one or more regions of a KLFtranscription factor, e.g., a transcription factor binding domain, orwhich bind to at least one of its endogenous transcription factorbinding sites. Peptide aptamer selection can be performed usingdifferent systems known in the art, including the yeast two-hybridsystem.

Also included are ADNECTINS™, AVIMERS™, and ANTICALINS that specificallybind to KLF transcription factor. ADNECTINS™ refer to a class oftargeted biologics derived from human fibronectin, an abundantextracellular protein that naturally binds to other proteins. See, e.g.,U.S. Application Nos. 2007/0082365; 2008/0139791; and 2008/0220049.ADNECTINS™ typically consists of a natural fibronectin backbone, as wellas the multiple targeting domains of a specific portion of humanfibronectin. The targeting domains can be engineered to enable anAdnectin™ to specifically recognize a therapeutic target of interest,such as a KLF transcription factor polypeptide, or a fragment thereof,e.g., a transcription factor binding domain, or at least one of itsendogenous transcription factor binding sites.

AVIMERS™ refer to multimeric binding proteins or peptides engineeredusing in vitro exon shuffling and phage display. Multiple bindingdomains are linked, resulting in greater affinity and specificitycompared to single epitope immunoglobulin domains. See, e.g., Silvermanet al., Nature Biotechnology. 23:1556-1561, 2005; U.S. Pat. No.7,166,697; and U.S. Application Nos. 2004/0175756, 2005/0048512,2005/0053973, 2005/0089932 and 2005/0221384.

Also included are designed ankyrin repeat proteins (DARPins), whichinclude a class of non-immunoglobulin proteins that can offer advantagesover antibodies for target binding in drug discovery and drugdevelopment. Among other uses, DARPins are ideally suited for in vivoimaging or delivery of toxins or other therapeutic payloads because oftheir favorable molecular properties, including small size and highstability. The low-cost production in bacteria and the rapid generationof many target-specific DARPins make the DARPin approach useful for drugdiscovery. Additionally, DARPins can be easily generated inmultispecific formats, offering the potential to target an effectorDARPin to a specific organ or to target multiple polypeptides/nucleicacids with one molecule composed of several DARPins. See, e.g., Stumppet al., Curr Opin Drug Discov Devel. 10:153-159, 2007; U.S. ApplicationNo. 2009/0082274; and PCT/EP2001/10454.

Certain embodiments include “monobodies,” which typically utilize the10th fibronectin type III domain of human fibronectin (FNfn10) as ascaffold to display multiple surface loops for target binding. FNfn10 isa small (94 residues) protein with a β-sandwich structure similar to theimmunoglobulin fold. It is highly stable without disulfide bonds ormetal ions, and it can be expressed in the correctly folded form at ahigh level in bacteria. The FNfn10 scaffold is compatible with virtuallyany display technologies. See, e.g., Batori et al., Protein Eng.15:1015-20, 2002; and Wojcik et al., Nat Struct Mol Biol., 2010; andU.S. Pat. No. 6,673,901.

Anticalins refer to a class of antibody mimetics, which are typicallysynthesized from human lipocalins, a family of binding proteins with ahypervariable loop region supported by a structurally rigid framework.See, e.g., U.S. Application No. 2006/0058510. Anticalins typically havea size of about 20 kDa. Anticalins can be characterized by a barrelstructure formed by eight antiparallel β-strands (a stable β-barrelscaffold) that are pairwise connected by four peptide loops and anattached α-helix. In certain aspects, conformational deviations toachieve specific binding are made in the hypervariable loop region(s).See, e.g., Skerra, FEBS J. 275:2677-83, 2008, herein incorporated byreference.

The therapeutic agents, e.g. binding agents, described herein whichinhibit the binding of a KLF transcription factor to its endogenoustranscription factor binding site(s), can be used in any of the methodsand compositions described herein.

Methods for Use

Embodiments of the present invention include methods of usingtherapeutic agents described herein (e.g., oligonucleotide decoys,binding agents), which inhibit or otherwise reduce binding of one ormore KLF transcription factors to its endogenous transcription bindingsite, and related compositions, to modulate the activity of one or moreKLF transcription factors. In particular embodiments, the one or moretranscription factors is selected from the group consisting of KLF1,KLF2, KLF3, KLF4, KLF5, KLF6, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13,KLF14, KLF15, KLF16 and KLF17.

The methods can be used, for example, to treat pain in a subject, tomodulate transcription of a gene present in a cell involved innociceptive signaling, to modulate transcription of a gene present in acell involved in perception of pain in a subject, and/or to modulatenociceptive signaling in a cell, for example, in a subject. Such methodscan be practiced in vitro, for instance, by contacting a cell with atherapeutic agent (e.g., oligonucleotide decoy) or related composition,or in vivo, for instance, by administering to a subject in need thereofa therapeutic agent (e.g., oligonucleotide decoy) or relatedcomposition. In particular embodiments, the therapeutic agent is anoligonucleotide decoy or population of oligonucleotide decoys, asdescribed herein.

Thus, certain embodiments include methods for treating pain in asubject, comprising administering to the subject a therapeuticallyeffective amount of a therapeutic agent, wherein the therapeutic agentinhibits binding of a transcription factor to its transcription bindingsite, and wherein the transcription factor is selected from the groupconsisting of KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF8, KLF9, KLF10,KLF11, KLF12, KLF13, KLF14, KLF15, KLF16 and KLF17. Also included aremethods of treating pain in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of oneor more oligonucleotide decoys described herein. In some embodiments,methods of preventing pain in a subject are provided, for example,prophylactic methods of treating or managing pain. Such methods compriseadministering to a subject in need thereof (e.g., a patient likely todevelop pain, e.g., post-operative pain) a therapeutically effectiveamount of an oligonucleotide decoy described herein.

Thus, in certain embodiments, an oligonucleotide decoy and/orpharmaceutical composition comprising the same is administered to asubject in need thereof, for example, such as an animal (e.g., a bird,mammal, primate, human patient), suffering from or expected to sufferfrom pain. Particular examples of pain include, but are not limited to,mechanical pain (e.g., mechanical hyperalgesia and/or allodynia),chemical pain, temperature pain, chronic pain, sub-chronic pain, acutepain, sub-acute pain, inflammatory pain, neuropathic pain, muscularpain, skeletal pain, post-surgery pain, radicular pain, back pain,arthritis pain, and/or diabetes pain. In certain embodiments, theoligonucleotide decoys and/or pharmaceutical compositions thereof areadministered to a patient, such as an animal, as a preventative measureagainst pain including, but not limited to, any one or more of theforegoing examples of pain. In some embodiments, the pain ispost-operative pain, chronic pain, inflammatory pain, neuropathic pain,muscular pain, and/or skeletal pain. In certain embodiments, theoligonucleotide decoys and/or pharmaceutical compositions thereof may beused for the prevention of one facet of pain while concurrently treatinganother symptom of pain.

In particular embodiments, the pain is chronic pain. In someembodiments, the pain is neuropathic pain, for example, chronicneuropathic pain and/or (chronic) neuropathic pain that is associatedwith inflammation (e.g., neuro-inflammation). In certain embodiments,the pain is associated with inflammation, for example, chronic painassociated with inflammation, chronic neuropathic pain associated withinflammation. In some embodiments, the pain is associated with thecentral nervous system and/or a visceral disorder. In some embodiments,the pain is post-surgical pain.

In some embodiments, the therapeutic agent (e.g., oligonucleotide decoy,population of oligonucleotide decoys, binding agent) or composition thatis administered to treat, manage, and/or prevent pain provides a bindingratio of KLF15/KLF9 equal to or less than about 0.8 or equal to orhigher than about 1.0 based on OD₄₅₀ values (or equivalent standardELISA measurement units) in a standard ELISA assay (see supra). Inspecific embodiments, the foregoing is used in the treatment ofneuropathic pain.

In particular embodiments, the therapeutic agent (e.g., oligonucleotidedecoy, population of oligonucleotide decoys, binding agent) orcomposition that is administered to treat, manage, and/or prevent painprovides a total transcription factor binding capacity to KLF6 and KLF9that is equal to or higher than a predetermined amount, for instance, anoptical density value of about 0.2 OD₄₅₀ (or comparable binding levelusing equivalent standard ELISA measurement units) in a standard ELISAassay (see supra). In some embodiments, the total transcription factorbinding capacity to KLF6 and KLF9 (as indicated by 1/(KLF6+KLF9)) isequal to or less than about 5, 4, 3, 2, 1, 0.5, 0.1, or less (includingall ranges and integers in between) based on OD₄₅₀ values (or anequivalent standard ELISA measurement unit, e.g., fluorescence) from astandard ELISA assay. In specific embodiments, the foregoing is used inthe treatment of pain or neuropathic pain associated with inflammation

Also included are methods for modulating transcription of a gene presentin a cell involved in nociceptive signaling and/or the perception ofpain in a subject, comprising administering to the cell atherapeutically effective amount of a therapeutic agent, wherein thetherapeutic agent inhibits binding of a transcription factor to itstranscription factor binding site, wherein the transcription factor isselected from the group consisting of KLF1, KLF2, KLF3, KLF4, KLF5,KLF6, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, KLF16 andKLF17. In some embodiments, the therapeutic agent includes one or moreoligonucleotide decoys described herein. In certain embodiments,modulation of transcription comprises suppressing or repressing geneexpression. In some embodiments, modulation of transcription comprisesstabilizing gene expression. In particular embodiments, modulation oftranscription comprises activating or inducing gene expression. Incertain embodiments, the gene is involved in nociceptive signaling.Genes involved in nociceptive signaling include, but are not limited to,genes encoding membrane proteins (e.g., ion channels, membranereceptors, etc.), soluble signaling molecules (e.g., intracellularsignaling molecules or neurotransmitters), synthetic enzymes (e.g.,neurotransmitter synthesis enzymes), and transcription factors. Specificexamples of such proteins include, but are not limited to, BDNF(regulated by KLF9), TGFB1 (regulated by KLF6), CDKN1A, JUN, GFAP(regulated by KLF15); and others such as BDKRB2, HTR3A, SCN9A, GRM5,NOS1, GCH1, CDK5R1, CACNA1B, P2XR3 and PNMT.

Some embodiments include methods for modulating nociceptive signaling ina cell, comprising administering to the cell a therapeutically effectiveamount of a therapeutic agent, wherein the therapeutic agent inhibitsbinding of a transcription factor to its transcription factor bindingsite, wherein the transcription factor is selected from the groupconsisting of KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF8, KLF9, KLF10,KLF11, KLF12, KLF13, KLF14, KLF15, KLF16 and KLF17. In some embodiments,the therapeutic agent includes one or more oligonucleotide decoysdescribed herein. In certain embodiments, modulation of nociceptivesignaling comprises suppressing or repressing nociceptive signaling. Insome embodiments, modulation of nociceptive signaling comprisesactivation of an inhibitor of nociceptive signaling. In particularembodiments, modulation of nociceptive signaling comprises increasingproteolytic degradation of a protein involved in nociceptive signalingin a cell. In certain embodiments, modulation of protein degradationcomprises stimulating proteasome function. In certain embodiments, theprotein is involved in nociceptive signaling. Proteins involved innociceptive signaling include, but are not limited to membrane proteins(e.g., ion channels, membrane receptors, etc.), soluble signalingmolecules (e.g., intracellular signaling molecules orneurotransmitters), synthetic enzymes (e.g., neurotransmitter synthesisenzymes), and transcription factors. Specific examples of such proteinsinclude, but are not limited to, BDNF (regulated by KLF9), TGFB1(regulated by KLF6), CDKN1A, JUN, GFAP (regulated by KLF15); and otherssuch as BDKRB2, HTR3A, SCN9A, GRM5, NOS1, GCH1, CDK5R1, CACNA1B, P2XR3and PNMT.

In certain embodiments, the cell of the various methods is provided invivo (e.g., in a subject suffering from pain or likely to suffer frompain). A cell provided in vivo can be located in different locationsincluding, but not limited to, a dorsal root ganglia and/or the spinalcord. In other embodiments, the cell of the various methods is providedin vitro (e.g., in a petri dish). The cell can be any cell involved innociceptive signaling, including, but not limited to, a neuron (e.g., apain neuron from dorsal root ganglia and/or the spinal cord or from thesympathetic nervous system), a glial cell, a tissue supportive cell(e.g., fibroblast), an immune cell, or a cell from a cell line (e.g., aPC12 cell).

In some embodiments, the oligonucleotide decoys and/or pharmaceuticalcompositions thereof are used in combination therapy with at least oneother therapeutic agent. Examples of other therapeutic agents includebut are not limited to one or more additional oligonucleotide decoys.The oligonucleotide decoy and/or pharmaceutical composition thereof andthe therapeutic agent can act additively or, more preferably,synergistically. In some embodiments, an oligonucleotide decoy and/or apharmaceutical composition thereof is administered concurrently with theadministration of another therapeutic agent, including anotheroligonucleotide decoy. In other embodiments, an oligonucleotide decoy ora pharmaceutical composition thereof is administered prior or subsequentto administration of another therapeutic agent, including anotheroligonucleotide decoy.

For administration to a subject in need thereof, the oligonucleotidedecoys and/or pharmaceutical compositions described herein may beadministered by any convenient route. Particular examples includeadministration by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.), and by oral administration. Administration canbe systemic or local. Various delivery systems are known in the art,including, e.g., encapsulation in liposomes, microparticles,microcapsules, capsules, etc., which can be used to administer acompound and/or pharmaceutical composition thereof. Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural/peridural, oral, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by inhalation or topically,particularly to the ears, nose, eyes, or skin. In certain embodiments,the oligonucleotide decoy is administered perineurally,epidurally/peridurally, intrathecally, or intradermally. In certainembodiments, more than one oligonucleotide decoy is administered to apatient. The preferred mode of administration is left to the discretionof the practitioner, and will depend in-part upon the site of themedical condition.

In specific embodiments, it may be desirable to administer one or moreoligonucleotide decoys locally to the area in need of treatment. Thismay be achieved, for example, and not by way of limitation, by localinfusion during surgery, topical application (e.g., in conjunction witha wound dressing after surgery), by injection, by means of a catheter,by means of a suppository, or by means of an implant, said implant beingof a porous, non-porous, or gelatinous material, including membranes,such as sialastic membranes, or fibers. In some embodiments,administration can be by direct injection at the site (e.g., former,current, or expected site) of pain.

In certain embodiments, it may be desirable to introduce one or moreoligonucleotide decoys into the nervous system by any suitable route,including but not restricted to intraventricular, intrathecal,perineural and/or epidural/peridural injection. Intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant.

The amount of oligonucleotide decoy that will be effective in thetreatment or prevention of pain in a patient will depend on the specificnature of the condition and can be determined by standard clinicaltechniques known in the art. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theamount of a oligonucleotide decoy administered will, of course, bedependent on, among other factors, the subject being treated, the weightof the subject, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician. Incertain embodiments, a single dose of oligonucleotide decoy comprisesabout 5 μg to about 15 mg, about 50 μg to about 7.5 mg, about 100 μg toabout 1 mg, about 250 μg to about 750 μg, or about 500 μg ofoligonucleotide decoy per kilogram (kg) of body weight.

In some embodiments, the dosage forms are adapted to be administered toa patient no more than twice per day, more preferably, only once perday. Dosing may be provided alone or in combination with other drugs andmay continue as long as required for effective treatment or preventionof pain.

Compositions and Kits

Certain embodiments include compositions, for example, pharmaceutical ortherapeutic compositions, comprising one or more therapeutic agents(e.g., oligonucleotide decoys, binding agents) described herein,optionally in combination with one or more pharmaceutically-acceptablecarriers (e.g., pharmaceutical-grade carriers).

The pharmaceutical compositions disclosed herein comprise atherapeutically effective amount of one or more therapeutic agents(e.g., oligonucleotide decoys), preferably, in purified form, togetherwith a suitable amount of a pharmaceutically-acceptable carrier, so asto provide a form for proper administration to a patient. Whenadministered to a patient, therapeutic agents such as oligonucleotidedecoys and pharmaceutically-acceptable carriers are preferably sterile.Examples of pharmaceutically-acceptable carriers include, but are notlimited to, saline, phosphate buffered saline (PBS), tris buffer, water,aqueous ethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. Water is a preferred vehicle whenoligonucleotide decoys are administered intravenously. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid vehicles, particularly for injectable solutions. Suitablepharmaceutically-acceptable carriers also include excipients such asstarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Pharmaceutical compositions, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.In addition, auxiliary, stabilizing, thickening, lubricating andcoloring agents may be used.

Pharmaceutical compositions may be manufactured by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients orauxiliaries, which facilitate processing of compounds disclosed hereininto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

Pharmaceutical compositions can take the form of solutions, suspensions,emulsions, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,aerosols, sprays, suspensions, or any other form suitable for use. Otherexamples of suitable pharmaceutical vehicles have been described in theart (see Remington's Pharmaceutical Sciences, Philadelphia College ofPharmacy and Science, 19th Edition, 1995).

Pharmaceutical compositions for oral delivery may be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions may contain one or more optional agents, forexample, sweetening agents such as fructose, aspartame or saccharin,flavoring agents such as peppermint, oil of wintergreen, or cherrycoloring agents and preserving agents, to provide a pharmaceuticallypalatable preparation. Moreover, when in tablet or pill form, thecompositions may be coated to delay disintegration and absorption in thegastrointestinal tract, thereby providing a sustained action over anextended period of time. Oral compositions can include standard vehiclessuch as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate, etc. Such vehicles arepreferably of pharmaceutical grade.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols(e.g., polyethylene glycol), oils, alcohols, slightly acidic buffersbetween pH 4 and pH 6 (e.g., acetate, citrate, or ascorbate at betweenabout 5 mM to about 50 mM), etc. Additionally, flavoring agents,preservatives, coloring agents, bile salts, acylcarnitines and the likemay be added.

For buccal administration, the compositions may take the form oftablets, lozenges, etc., formulated in conventional manner. Liquid drugformulations suitable for use with nebulizers and liquid spray devicesand EHD aerosol devices will typically include a compound with apharmaceutically acceptable vehicle. In some aspects, thepharmaceutically acceptable vehicle is a liquid such as alcohol, water,polyethylene glycol or a perfluorocarbon. Optionally, another materialmay be added to alter the aerosol properties of the solution orsuspension of compounds. In some aspects, the material is liquid such asan alcohol, glycol, polyglycol or a fatty acid. Other methods offormulating liquid drug solutions or suspension suitable for use inaerosol devices are known to those of skill in the art (see, e.g.,Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611).A compound may also be formulated in rectal or vaginal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, a compound may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, acompound may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

An oligonucleotide decoy may be included in any of the herein-describedformulations, or in any other suitable formulation, as apharmaceutically acceptable salt, a solvate or hydrate. Pharmaceuticallyacceptable salts substantially retain the activity of the parentcompound and may be prepared by reaction with appropriate bases or acidsand tend to be more soluble in aqueous and other protic solvents thanthe corresponding parent form.

In some instances, liposomes may be employed to facilitate uptake of theoligonucleotide decoys into cells, for example, in vitro or in a subject(see, e.g., Williams, S. A., Leukemia 10(12):1980-1989, 1996;Lappalainen et al., Antiviral Res. 23:119, 1994; Uhlmann et al.,Chemical Reviews, Volume 90, No. 4, 25 pages 544-584, 1990; Gregoriadis,G., Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp.287-341, Academic Press, 1979). Hydrogels may also be used as vehiclesfor oligonucleotide decoy administration, for example, as described inWO 93/01286. Alternatively, the oligonucleotide decoys may beadministered in microspheres or microparticles. (See, e.g., Wu, G. Y.and Wu, C. H., J. Biol. Chem. 262:4429-4432, 30 1987). Alternatively,the use of gas-filled microbubbles complexed with the oligonucleotidedecoys can enhance delivery to target tissues, as described in U.S. Pat.No. 6,245,747. Sustained release compositions may also be used. Thesemay include semipermeable polymeric matrices in the form of shapedarticles such as films or microcapsules.

Oligonucleotide decoys can be introduced into cells using art-recognizedtechniques (e.g., transfection, electroporation, fusion, liposomes,colloidal polymeric particles and viral and non-viral vectors as well asother means known in the art). The method of delivery selected willdepend at least on the oligonucleotide chemistry, the cells to betreated and the location of the cells and will be apparent to theskilled artisan. For instance, localization can be achieved by liposomeswith specific markers on the surface to direct the liposome, directinjection into tissue containing target cells, specificreceptor-mediated uptake, or the like.

As known in the art, oligonucleotide decoys may be delivered using,e.g., methods involving liposome-mediated uptake, lipid conjugates,polylysine-mediated uptake, nanoparticle-mediated uptake, andreceptor-mediated endocytosis, as well as additional non-endocytic modesof delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (see, e. g., Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44:35-49, incorporated by reference inits entirety).

In certain embodiments, one or more oligonucleotide decoys are providedin a kit. In certain embodiments, the kit includes an instruction, e.g.,for using said one or more oligonucleotide decoys. In certainembodiments, said instruction describes one or more of the methods ofthe present invention, e.g., a method for preventing or treating pain, amethod of modulating gene expression in a cell, a method for modulatingnociceptive signaling in a cell, a method for modulating proteindegradation in a cell, etc. In certain embodiments, the oligonucleotidedecoys provided in a kit are provided in lyophilized form. In certainrelated embodiments, a kit that comprises one or more lyophilizedoligonucleotide decoys further comprises a solution (e.g., apharmaceutically-acceptable saline solution) that can be used toresuspend one or more of the oligonucleotide decoys.

The following examples are intended to illustrate but not to limit theinvention. Each of the patent and non-patent references referred toherein is incorporated by reference in its entirety.

EXAMPLES Example 1 Targeting the KLF Family for the Treatment of Pain

Oligonucleotide decoys targeted against members of the Krüppel-likefamily of transcription factors (KLFs) were designed, characterized forKLF-binding, and tested in animal models of neuropathic andneuro-inflammatory pain.

Cross-analysis of the KLF binding patterns, efficacy amplitude, andduration across two separate neuropathic and neuro-inflammatory painmodels (described below) showed that the oligonucleotide decoys TFD16(GATCCTTTGCCTCCTTCGATCCTTTGCCTCCTTCAAG; SEQ ID NO:37) and TFD17(GGTGTTTGGGAGAGCTTTGGGAGGATACG; SEQ ID NO:38) were effective in bothmodels and acted through inhibiting KLF6, 9 and/or 15 (data not shown).These data showed that efficacy for reducing chronic neuropathic pain iseffective through the combined inhibition of KLF9 and KLF15, andefficacy for reducing chronic neuro-inflammatory pain is effectivethrough the combined inhibition of KLF6 and KLF9.

Consequently, TFD16 and TFD17 were selected as sequence matrices togenerate additional oligonucleotide decoy sequences with complementaryKLF6, 9, and 15 binding patterns. Based on this analysis, theoligonucleotide decoys in Table 2 (supra) were prepared tested for KLFbinding, and those in Table E1 (below) were further tested in animalmodels of pain, as described below.

TABLE E1 SEQ ID Name Sequence (5′ to 3′) NO: 16.6.2CCTTTGCCTCCTTCGCCTCCTTCAA 25 16.6.5 ATCCTTCGCCTCCTTCAA 28 16.9ATCCTTTGCCTTTGCCTCCTTCAA 12 17.1 GCTTTGGGAGGATAC 15 17.5TGTTTGGGAGAATCCTCCCAAAGC 19 17.9 TGTTTGGGAGAGGGAGGATAC 23 TFD3GCGCACCCCAGCCTGGCTCACCCACGCG 36 TFD16GATCCTTTGCCTCCTTCGATCCTTTGCCTCCTTCAAG 37 TFD17GGTGTTTGGGAGAGCTTTGGGAGGATACG 38

ELISA Assay. KLF binding of the oligonucleotide decoys was measuredusing a customized version of an SP1 commercial ELISA kit (SP1 ELISAKit, catalogue number EK-1090, Affymetrix). Briefly, biotin-decoy probes(12.8 pmoles/well) were incubated with 15 μg of nuclear protein extractscontaining KLF transcription factors from either (a) HELA cells: forKLF1-6, 8-14, and 16-17 detection (catalogue #36010, Active motif, CA)or (b) HEK290: for KLF15 detection (catalogue # 36033, Active motif,CA). For KLF 7 detection, 0.5 and 1 μg of a recombinant human KLF7protein was utilized (Novus, CA, catalogue # NBP2-23176).

The processing of the decoy probe-protein mix was performed according tothe ELISA kit supplier: the mix was loaded on streptavidin-coated96-well plates, and the quantity of captured KLF measured with anantibody-based colorimetric detection (anti-rabbit secondary antibodyconjugated to HRP) in a microplate reader (OD₄₅₀ nm). When increasingconcentration of competing, non-biotinylated decoys were added to thebinding mix reaction, a reduction of transcription factor binding to thebiotinylated probe is a demonstration of binding specificity. All datawere corrected against background signal measured internally for eachELISA run, and all testing steps and testing conditions werestandardized according to the kit supplier's recommendation, includingdetection time with the detection buffer (i.e., 5.2 min determined asoptimal for this assay), to ensure appropriate comparison of brute OD₄₅₀binding values between ELISA runs.

For the ELISA assays, single strands of each decoy were manufactured byInvitrogen (CA), re-suspended in 100 μM stock solution in TE pH 8, NaCl50 μM, and annealed in 4 μM working solutions as follow: (a) decoy mix(100 μl): 4 μL sense strand (100 μM)+4 μl antisense strand (100 μM)+89.5μl pH 8+2.5 μl NaCl (1.94 M); (b) annealing: 7 min at 95° followed byslow cooling at RT for 1 h before use or storage at −20° C.

Binding specificity was assessed by measuring binding signal linearity,reduction of binding with free competitor KLF decoy, and by the lack ofKLF binding to mutant decoys (See Table E2 below).

TABLE E2 Mutant decoys SEQ ID Name Sequence (5′ to 3′) NO: MUT1 SATGCAGGAGAAAGATTGGCGTAGTATCTACTAG 39 MUT1 ASCTTCATGATTTTATTGCTTTCAAAATCCAAAAT 40 MUT2 SGTTATGCGTTTGTAGATGCTTTCGTTATAG 41 MUT2 AS CTATTTCGAAACGATCTACATTGGCATAAC42

Rabbit primary KLF antibodies were obtained from commercially-availablesources, and the secondary anti-rabbit antibody conjugated to HRP usedfor the KLF assay was the antibody provided in the ELISA kit (dilution1:200).

In vivo Efficacy Studies. The materials and methods for the animalmodels of pain are described below.

Animals.

Sprague-Dawley rats, 250-300 g, males, Harlan Industries (Livermore,Calif.).

Test and Control Articles.

For animal testing, oligonucleotide decoys were manufactured by TrilinkBiotechnologies (CA) and formulated as 10 mM or 15 mM stock solutions(Tris-pH 7.5, CaCl₂). Each decoy was prepared for 20 μL injections atthe appropriate concentration for the selected dose delivery.Oligonucleotide decoy and vehicle controls (Tris-10 mM, 140 mM NaCl, pH7.5) were provided to the testing site in a blinded fashion and inready-to-use vials.

Spared Nerve Injury (SNI) Model.

Anaesthesia was induced with 2% isoflurane in O₂ at 2 L/min andmaintained with 0.5% isofluorane in O₂. Rats were then shaved andaseptically prepared for surgeries. Spared nerve injury was done basedon the method described by Decosterd et al (Decosterd and Woolf, 2000).Briefly, skin and fascia of left thigh were incised, two heads of m.biceps femoris spread, and 3 terminal branches of sciatic nerve exposed.Tibial and comon peroneal were tightly ligated, dissected distally toligation, and 2-3 mm of nerve trunk was removed. The sural branch wasleft intact. The wound was closed in a layered fashion.

Chronic Constriction Injury (CCI) Model.

Following the Chronic Constriction Injury model (Bennett and Xie, 1988),the right common sciatic nerve was exposed at the level of the middle ofthe thigh by blunt dissection through the biceps femoris. Proximal tothe sciatic's trifurcation, about 12 mm of nerve was freed of adheringtissue and four ligatures were tied loosely around it with about 1 mmspacing. The length of nerve thus affected was 6-8 mm long. Care wastaken to tie the ligatures such that the diameter of the nerve was seento be just barely constricted when viewed with 40× magnification. Thedesired degree of constriction retarded, but did not arrest, circulationthrough the superficial epineural vasculature and sometimes produced asmall, brief twitch in the muscle surrounding the exposure. The incisionwas closed in layers.

Mechanical Hypersensitivity.

Pain was measured as mechanical hypersensitivity using repetitive vonFrey filament testing. Briefly, von Frey filaments (1-4-6-8-10-10-26 g)were used to test for the responsiveness to mechanical stimulation ofthe hind paw. Animals were habituated on a mesh floor 1 hour prior totesting and five applications of each filament was applied. For eachapplication, the hair was pressed perpendicularly against the paw withsufficient force to cause slight bending, and held for approximately 1-2seconds. A positive response was noted if the paw was sharply withdrawn.Flinching immediately upon removal of the hair was also considered apositive response. Stimuli were presented successively following thepattern described above. Animals were tested at baseline just prior tosurgery and at determined time-points pre- and post-injections.

Blinding & Randomization.

All experiments were performed blinded. The testing sites receivedblinded vials and the blinding code being was broken after testing wascompleted.

Randomization was performed on POD14 after the baseline pain testing andbefore the dosing. For each tested cohort, animals were distributed ingroups of 2 to 3 rats so mean POD14 von Frey values were as close aspossible across the testing groups, targeting within 15% of each otherif the values permits. Once animals were distributed into groups, theattribution of solution treatment to groups was at the discretion of theexperimenter.

Pre-defined Inclusion & Exclusion Criteria.

Animals with von Frey values 5 at the day of the first injection (i.e.,POD14) was excluded from results analysis. The von Frey value of 5 isbased on internal historical data across multiple testing sites whererats can reach this value in basal condition, pre-surgery and istherefore a threshold for the absence/presence of model-inducedhypersensitivity. If the average of mechanical hypersensitivity valuesof vehicle-treated were reduced by 50% or more during the first weekfollowing injection, the cohort was excluded on the ground that the painmodel did not perform appropriately.

Intrathecal Delivery.

Oligonucleotide decoys were delivered intrathecally. Sprague-Dawley ratswere anesthetized with 2% isoflurane, their backs shaved and preparedwith Betadine. The rat then was placed on a bottle to keep the backarched. A 17 G ½ needle was slid rostrally along left side of the L6vertebra level transverse process until it reached the L5 vertebralevel. The needle was then inserted between L5 and L6 until theintrathecal space was reached as indicated by tail twitch. 20 μL ofdecoy or vehicle were then injected intrathecally (IT). Depending on thestudy, rats received either a single IT injection once at POD14following surgery, or once at POD14 and once at POD17.

Statistical Analysis.

Non-parametric Student T-test followed by a T-Welsh analysis for unevenvariance correction was used to analyze individual time-points and datadistribution comparison between experimental conditions.

The results from the KLF-binding ELISA analysis and CCI and SNI animalmodels of pain with single dosing level of decoys are shown in FIGS.1-3B. FIG. 1 shows the KLF binding characteristics of theoligonucleotide decoys from Tables 2 and E1, relative to independentcontrol KLF decoys (highlighted in gray; see, e.g., Shields and Yang,1998; Matsumuto et al., 1998). Binding values to KLF6, KLF9, and KLF15are presented as mean and SEM OD₄₅₀ values from the in vitro ELISAbinding assay described in Example 1. The corresponding N is alsolisted. The efficacy for treating neuropathic and/or neuro-inflammatorypain is presented as percentage (%) of pain reduction relative tocontrol during the testing period of the corresponding animal studies.

FIGS. 2A-B show the efficacy of the tested oligonucleotide decoys in theSNI model of chronic neuropathic pain, and FIGS. 3A-B show the efficacyof the tested oligonucleotide decoys in the CCI model of chronicneuro-inflammatory pain.

A detailed meta-analysis of the combined in vivo efficacy and in vitrobinding results for all of the oligonucleotide decoys tested in vivo wasconducted to characterize the relationship between the KLF bindingpattern and the efficacy of the oligonucleotide decoys. FIG. 4A-C showthe efficacy level of the oligonucleotide decoys for treating chronicneuropathic pain in relation to their ratio of KLF15/KLF9 binding (4A),coefficients of linear correlation between the efficacy for treatingchronic neuropathic pain and the binding parameters to KLF6, KLF9 andKLF15 (4B), and a linear regression of efficacy levels in relation toKLF15/KLF9 binding ratios (4C).

Similarly, FIGS. 5A-C show the efficacy level of the oligonucleotidedecoys for treating chronic neuro-inflammatory pain in relation to theircombined binding to KLF6, KLF9, and KLF15 (5A), coefficients of linearcorrelation between the efficacy for treating chronic neuro-inflammatorypain and the binding parameters to KLF6, KLF9 and KLF15 (5B), and alinear regression of efficacy level in relation total transcriptionfactor binding capacity to KLF6 and KLF9, as indicated by the1/(KLF6+KLF9) binding ratio (5C).

FIG. 6 shows the differential and superior pattern of efficacy of thedecoys from the invention relative to control KLF consensus decoys fromthe literature (TFDC1, TFDC2, and TFD3), across complementary etiologiespain, from neuropathic (Y-axis) to pain including inflammatorycomponents (X-axis).

FIG. 7 shows a plot of the 1/(KLF6+KLF9) binding ratio, which isindicative of the efficacy for treating neuro-inflammatory pain (thelower, the more efficacy), and the KLF15/KLF9 binding ratio, which isindicative of the efficacy for treating neuropathic pain (the higher,the more efficacy), for the oligonucleotide decoys in Table 2. Eachnumber in the X-axis corresponds to an individual decoy.

To further characterize the therapeutic profile of the 16.6.5oligonucleotide decoy, dose response studies were performed in the SNIand CCI animal models. FIGS. 8A-B show the robust and long-lastingefficacy of ascending dose levels from 50 to 300 nmoles in these twoanimal models of pain.

Altogether, these studies not only identify the family of KLFtranscription factors as targets of therapeutic relevance for treatingpain, but also identify a set of oligonucleotide sequences with uniquebinding profile to KLF transcription factors, relative to previouslydescribed KLF sequences, which are associated with a unique and robustpotential for treating in vivo pain across multiple etiologies.

The invention claimed is:
 1. A population of oligonucleotide decoys, theoligonucleotide decoys comprising a combination of at least twotranscription factor binding sites, wherein each transcription factorbinding site binds to at least one transcription factor selected fromthe group consisting of KLF6, KLF9, and KLF15, wherein: the populationof oligonucleotide decoys provides a transcription factor binding ratioof KLF15/KLF9 equal to or less than about 0.8 or equal to or higher thanabout 1.0 in a standard ELISA assay; or the population ofoligonucleotide decoys provides a total transcription factor bindingcapacity to KLF6 and KLF9 that is equal to or higher than an opticaldensity value of about 0.2 OD₄₅₀ in a standard ELISA assay.
 2. Thepopulation of oligonucleotide decoys of claim 1, wherein theoligonucleotide decoys are about 15 to about 35 base pairs in length. 3.The population of oligonucleotide decoys of claim 1, wherein theoligonucleotide decoys comprises a first transcription factor bindingsite and a second transcription factor binding site, and wherein thefirst and the second transcription binding sites overlap.
 4. Thepopulation of oligonucleotide decoys of claim 1, wherein theoligonucleotide decoys have a first transcription factor binding site, asecond transcription factor binding site, and a third transcriptionfactor binding site, and wherein the first, second, and thirdtranscription factor binding sites overlap.
 5. The population ofoligonucleotide decoys of claim 4, wherein the first transcriptionfactor binding site binds to KLF6, the second transcription factorbinding site binds to KLF9, and the third transcription factor bindingsite binds to KLF15.
 6. The population of oligonucleotide decoys ofclaim 3, wherein the first transcription factor binding site binds toKLF9 and the second transcription factor binding site binds to KLF15. 7.The population of oligonucleotide decoys of claim 3, wherein the firsttranscription factor binding site binds to KLF9 and the secondtranscription factor binding site binds to KLF6.
 8. A pharmaceuticalcomposition comprising a population of oligonucleotide decoys of claim 1and a pharmaceutically acceptable carrier.
 9. A kit comprising apopulation of oligonucleotide decoys of claim 1, and optionally aninstruction for using said oligonucleotide decoy.
 10. A method formodulating nociceptive signaling in a cell comprising administering tothe cell an effective amount of a population of oligonucleotide decoysof claim
 1. 11. A method for treating pain in a subject comprisingadministering to the subject a therapeutically effective amount of apopulation of oligonucleotide decoys of claim
 1. 12. The method of claim11, wherein the pain is a chronic pain.
 13. The method of claim 11,wherein the pain is neuropathic pain.
 14. The method of claim 11,wherein the pain is associated with inflammation.
 15. The method ofclaim 11, wherein the pain is associated with the central nervous systemor visceral disorder.
 16. A method for modulating nociceptive signalingin a cell comprising administering to the cell a therapeuticallyeffective amount of a therapeutic agent, wherein the therapeutic agentinhibits binding of a transcription factor to its transcription factorbinding site, wherein the transcription factor is selected from thegroup consisting of KLF6, KLF9, and KLF15, wherein the therapeutic agentprovides a binding ratio of KLF15/KLF9 equal to or less than about 0.8or equal to or higher than about 1.0 in a standard ELISA assay, or thetherapeutic agent provides a total transcription factor binding capacityto KLF6 and KLF9 that is equal to or higher than an optical densityvalue of about 0.2 OD₄₅₀ in a standard ELISA assay.
 17. A method fortreating pain in a subject comprising administering to the subject atherapeutically effective amount of a therapeutic agent, wherein thetherapeutic agent inhibits binding of a transcription factor to itstranscription binding site, wherein the transcription factor is selectedfrom the group consisting of KLF6, KLF9, and KLF15, wherein: thetherapeutic agent provides a binding ratio of KLF15/KLF9 equal to orless than about 0.8 or equal to or higher than about 1.0 in a standardELISA assay; or the therapeutic agent provides a total transcriptionfactor binding capacity to KLF6 and KLF9 that is equal to or higher thanan optical density value of about 0.2 OD₄₅₀ in a standard ELISA assay.18. The method of claim 17, wherein the pain is neuropathic pain.